Breast cancer metastasis to the lungs, bones, liver and brain is the fatal stage in the most common form of cancer diagnosed annually in women in the US. Tumor hypoxia and necrosis are tightly correlated with metastasis and poor prognosis in breast cancer, although the molecular basis of this correlation is not well defined. BNIP3 is a hypoxia- inducible regulator of cell death and loss of BNIP3 activity increased the metastatic potential of breast tumor cell lines in mouse xenografts. Mechanistic studies from our lab have indicated a critical role for BNIP3 in promoting autophagy and limiting necrosis in mammary tumor cells in culture. The current work will extend our molecular insight into the functions of BNIP3 by examining the molecular mechanism underlying the role of BNIP3 in targeting mitochondria for autophagy and how this contributes to the adaptive response of breast tumor cells to hypoxia (Aim 1). Making use of primary tumor arrays, we will examine the statistical significance of BNIP3 levels and altered sub-cellular localization for predicting progression of human breast cancer (Aim 2). Using structure-function analyses, we examine molecular mechanisms that may explain BNIP3 inactivation during breast cancer progression (Aim 2). The proposed work will also use state-of-the-art imaging techniques to monitor BNip3 expression during mammary tumor progression and metastasis in mouse models of breast cancer and to monitor the effect of BNip3 gene knockout on the incidence and latency of breast tumor metastasis to the lungs in cancer-prone mice (Aim 3). This work has clinical significance by identifying BNIP3 as a putative marker of breast cancer progression and defining a novel role for BNIP3 as a metastasis suppressor. PUBLIC HEALTH RELEVANCE: Metastasis is the last and most deadly step in the progression of human cancers and in the case of breast cancer the spread of tumor cells to the lungs, bones, liver and brain is a poor prognosis for survival. The significance of the current work lies in defining inactivation of BNIP3 as a marker of tumor progression, characterizing its molecular function and mechanism of inactivation in primary human breast cancer, and in determining whether it functions as a metastasis suppressor. Using data from primary human breast cancers and from mouse models to determine whether BNIP3 plays a role in preventing breast cancer metastasis, our work aims to determine whether BNIP3 might be useful as a molecular marker of breast cancer progression and if intervention to prevent BNIP3 inactivation is likely to reduce the incidence of breast cancer metastasis.